伤寒沙门菌fljB∷lacZ变异株的制备

目的：为研究伤寒沙门菌z66鞭毛抗原编码基因fliB的表达调节机制，建立该fljB基因的β-半乳糖苷酶基因（1acZ）插入变异株。方法：采用自杀质粒介导的同源重组方法进行制备。设计加接茚nI和SalI的特异性引物，用PCR扩增获得舻基因的上、下游同源性DNA片段，并与用Kpn I和Sal I酶切pSV-β-半乳糖苷酶载体（pSV-β-galactosidase vector）的片段定向连接，将连接片段克隆至自杀质粒pGMBl51，再通过电击法将重组质粒转入伤寒沙门菌，在含X-Gal的蔗糖平板上筛选获得同源重组的变异株。结果：经筛选获得阳性克隆，经PCR及序列分析证实,fljB基因中的763bp被3550bp的lacZ片段替代。结论：成功构建伤寒沙门菌flj∷lacZ突变株，为进一步研究fljB基因表达调节机制奠定了基础。

Construction of a fljB∷lacZ mutant of Salmonella enterica serovar Typhi

Objective： To study the mechanisms of expressional mutant strain of Salmonella enterica serovar Typhi was constructed. regulation of fljB： z66, the fljB∷lacZ Methods： The mutant strain was prepared by the method of homologous recombination mediated by suicide plasmid. The special primers with Kpn I and Sal I sites respectively were designed, the upper-and down-stream homologous fragments of the fljB gene were amplified by PCR, a lacZ cassette from the pSV-β-galactosidase vector was linked with the upper-and doun-stream homologous fragments respectively. The resulting product was inserted via Sma I sites into the suicide plasmid pGMB151. S. enterica serovar Typhi were transformed with recombinant plasmid by electroporation transformation, selected in the sucrose plate containing X-Gal. Homologous recombinants were screened by PCR. Results： A 3 550 bp insertion in fljB gene was confirmed instead of 763 bp deletion by PCR and sequencing analysis. Conclusion： The mutant with fljB：：lacZ have been successfully constructed and it was a foundation to study the expression and regulation mechanism of fljB gene in S. Typhi.